Sulfation at Glycopolymer Side Chains Switches Activity at the Macrophage Mannose Receptor (CD206) In Vitro and In Vivo

The mannose receptor (CD206) is an endocytic receptor expressed by selected innate immune cells and nonvascular endothelium, which plays a critical role in both homeostasis and pathogen recognition. Although its involvement in the development of several diseases and viral infections is well established, molecular tools able to both provide insight on the chemistry of CD206-ligand interactions and, importantly, effectively modulate its activity are currently lacking. Using novel SO4-3-Gal-glycopolymers targeting its cysteine-rich lectin ectodomain, this study uncovers and elucidates a previously unknown mechanism of CD206 blockade involving the formation of stable intracellular SO4-3-Gal-glycopolymer–CD206 complexes that prevents receptor recycling to the cell membrane. Further, we show that SO4-3-Gal glycopolymers inhibit CD206 both in vitro and in vivo, revealing hitherto unknown receptor function and demonstrating their potential as CD206 modulators within future immunotherapies.

3-O-sulfo-galactose was synthesized following the method reported by Kiessling and coworkers, 6, 7 with some modifications.
To a solution of 2'-azidoethyl-O-β-D-galactopyranoside (1.00 g, 4.02 mmol) in MeOH (10 mL), EtOAc (5 mL) and solid phenylboronic acid (490 mg, 4.02 mmol) were added. A Dean-Stark apparatus, pre-filled with EtOAc, was set up and the reaction mixture was stirred under reflux for 2 h. The reaction solution was then concentrated to ca. 5 mL, a fresh portion of MeOH/EtOAc (10 mL) and solid dibutyltin oxide (1.26 g, 4.42 mmol) were added, and the reaction mixture was refluxed using the Dean-Stark apparatus for further 2 h. The solvent was then removed under reduced pressure, and the resulting residue was dissolved in anhydrous DMF (5 mL) and added of trimethylamine sulfur trioxide (700 mg, 5.03 mmol). After stirring overnight at room temperature, MeOH (5 mL) was added to stop the reaction, the solvent removed under reduced pressure, and the residue added of a second portion of MeOH (5 mL) and stirred. After 10 min, the crude product was precipitated in Et2O, redissolved in methanol and precipitated again in Et2O to remove any residual traces of DMF. The resulting precipitate was added of MeOH (5 mL), diluted with water (10 mL) and extracted with Et2O (3 x 10 mL).
The aqueous layer was then added of anionic exchange resin (Dowex Na + , previously extensively washed with methanol). The mixture was stirred for 15 min, then the resin was removed by filtration. The aqueous solution was freeze-dried, and the resulting residue was
Synthesis of (propargyl methacrylate)187 (PMA187). Degree of polymerization (DP) and Mn,NMR were estimated by comparing the signals of the polymer chain-end at 5.3 (CHO) and 6.6 (CHvinyl) ppm, and those of the propargyl ester repeating units at 4.6 (CH2O) and 2.5 (C≡CH) ppm.

Synthesis of Oregon Green-labelled glycopolymers
Glycopolymers were prepared by clicking Oregon Green and sugar azides to appropriate poly(propargyl methacrylate) polymeric precursors by CuAAC. 2 As an example, the preparation of Oregon Green-tagged (S100%)32 (S32) is described below.
A solution of poly(propargyl methacrylate) (DP 32, 300 mg, 2.42 mmol of clickable alkyne units), Oregon Green azide (11 mg, 0.024 mmol) and bipyridine (152 mg, 0.967 mmol) in DMF (15 mL) were degassed by nitrogen bubbling for 15 min. CuBr (I) (69.5 mg, 0.484 mmol) was then added to the reaction mixture under a positive flow of nitrogen, and the solution was bubbled with nitrogen for further 15 minutes. The deep purple solution was stirred at room temperature for 3 days. The reaction was monitored by SEC with visible (λ = 496 nm) and RI detection. A 5 mL aliquot was withdrawn, and to this 3-O-sulfo-2'-azidoethyl-Ogalactopyranoside (6) (319 mg, 0.968 mmol) was added and the solution degassed for 15 min under nitrogen. A solution of sodium ascorbate (32 mg, 0.16 mmol) in water (100 µL) was added to the mixture with a deoxygenated syringe, and the reaction was stirred at room temperature for further 2 days. The glycopolymer was precipitated in THF, isolated by centrifugation, re-dissolved in water, transferred into a dialysis membrane (MWCO 3.5 kDa) and dialyzed in the dark against aqueous EDTA, then DI water for 3 days. The polymer aqueous solution was then freeze-dried to give (S100%)32 (S32) as a pink-orange solid.
For the synthesis of glycopolymers with 66 or 33% of mannosylated or galactose 3-O-sulfate sugars, the procedure is analogous to that described above, except that after clicking of the Oregon Green azide, mannose and sulfated galactose azides were first added to the reaction mixture according to the required molar ratio (alkyne units/sugar 100:66 or 100:33) and stirred at room temperature. After 3 days an excess of galactose azide, to give a total (sugar azide)/alkyne units molar ratio of 1.5:1, was added and the mixture left to react for further 3 days, to react all the clickable units available. The synthetic protocol followed in this work diverges slightly from that reported previously by Haddleton and us, 9 in that the sugar and fluorescent azides were added sequentially over time, rather than used all together from the beginning of the click reaction. This was done because in our previous study we only utilized 2'-azidoethyl-O-galacto-and 2'-azidoethyl-O-mannopyranoside, which for this 'click' reaction one can assume they are so similar that their kinetics of reaction with poly(propargyl methacrylate) are almost identical. Hence a copolymer with a specific relative content of these two sugar moieties could be prepared by simply mixing the two sugar azide precursors in the same molar ratio (co-clicking). In this work, however, 3-O-sulfo-2'-azidoethyl-Ogalactopyranoside is larger than the corresponding Man and Gal azides, and is also negatively charged, hence the assumption that it would react with the same rate as the other azides may not be valid. Therefore, here functionalisation of clickable poly(propargyl methacrylate) was carried out by adding the required sugar azides in a sequential manner over time, as described above.
The synthesis of Nile blue-labelled polymer (M32-Nile Blue) was performed under identical conditions, but with using Nile blue azide instead of Oregon Green azide. Molar fraction of each repeating unit (e.g. 0.33 indicates that 33% of polymer repeating units contain that specific sugar molecule, 0.01 that 1% of repeating units are functionalised with a fluorophore (Fluor.), etc). Molar fractions are those expected based on the relative amount of reagents used in the 'click' reaction feed. b) All glycopolymers are fluorescently tagged with Oregon Green, except for M32-Nile Blue, which was tagged with Nile Blue. c) Calculated by multiplying the degree of polymerisation (DP) of the relevant clickable poly(propargyl methacrylate) precursor (either 32 or 187) by the molecular mass of the clicked sugar repeating units, + the molecular weight of ATRP initiator used to prepare the clickable precursor: Mn,theor = (DP x MWsugar repeating units)+ MWATRP initiator. d) Obtained from SEC analysis using DMF+0.1% LiBr as the mobile phase (PMMA standards). e) Obtained from SEC analysis using PBS as the mobile phase (PEG standards). For clarity, glycopolymers where all repeating units (100%) present the same monosaccharide repeating unitse.g. (G100%)32, (M100%)32, etc.were renamed with a simpler alternative polymer code, as indicated in the alt. code column.  Figure S3. Overlay of 13 C NMR spectra of glycopolymers (DP 32) with different galactose-3sulfate content, in DMSO-d6. For this subfamily of CD206 ligands, 13 C NMR allows better characterization of glycopolymers with different galactose (•):SO4-3-galactose (•) polymer ratios than 1 H NMR, due to the signal broadening observed in the 1 H NMR spectra as the galactose-3-sulfate content increases ( Figures S33-35, and S38).

Synthesis of Texas Red-tagged gelatin (TR-Gelatin)
Texas Red-tagged gelatin was prepared following the procedure described by Hummert et al.  exchanges. After complete removal of the unreacted Texas Red, the solution was freeze-dried and stored at -20°C.
Bone marrow-derived macrophages (BMDM) from WT and CD206 -/-C57BL/6 mice were prepared from femurs and tibias using L929 cells conditioning media ( Figure S6. CHO and CD206 + -CHO cells viability at 2, 24, and 48 h, following incubation with SO4-3-Gal glycopolymers S32 and S187 for 0.5 or 2 h: LDH assay. CHO and CD206 + -CHO cells were pre-incubated with S32 or S187 for (A) 30 min, or (B) 2 h. Glycopolymer-containing culture media were replaced with fresh media, and LDH assay was carried out after 2, 24, or 48 h. Concentrations of S32 and S187 are expressed in µM of sugar repeating units. Data shown are expressed as cell viability (%), and are the average of two independent experiments performed in triplicate.

Uptake inhibition/co-incubation experiments: after initial incubation with G32 or S32, M32-Nile
Blue (4.04 µL, 16.5 mM sugar units) was added to the wells without removing the initial glycopolymers-containing medium. Cells were co-incubated with M32-Nile Blue and G32/S32 containing medium for further 2 h at 37°C in the dark, and then treated as described above.
Cells treated with only M32-Nile Blue for 2 h, without pre-incubation with G32 or S32 glycopolymers, were used as positive controls, and their MFI readings set as 100% of ligand uptake. Fluorescence was assessed by FACS analysis on FL1 (λ = 525 nm, Oregon Green detection) and FL4 (λ = 675 nm, Nile blue detection). Fluorescence of samples was reported as % relative to that of the positive control.   Figure 3 of the manuscript.

Glycopolymers internalization and intracellular trafficking: confocal microscopy studies
These experiments were carried out to confirm that glycopolymers were indeed internalized by CD206 + -cells and not simply associated to CD206 at the cell membrane, and to follow intracellular trafficking of glycopolymers after CD206-mediated endocytosis. Accordingly, 2x10 5 CD206 + -CHO cells were allowed to adhere to 10x10 mm glass slide (pre-treated with • To monitor glycopolymer internalization: after gentle washing with PBS (3 x 1 mL) cells were fixed with 4% paraformaldehyde in PBS for 10 minutes at ambient temperature, samples were rinsed with PBS (3 x 1 mL) and directly stained with 1 µg mL -1 HOECHST solution in PBS for 15 min at room temperature.
• To follow glycopolymer trafficking: after gentle washing with PBS (3 x 1 mL) cells were either immediately fixed with 4% paraformaldehyde in PBS for 10 minutes at ambient temperature or incubated for further 2 h with complete medium before fixation. Samples were rinsed with PBS (3 x 1 mL) and permeabilized in 0.5% Tween 20 with 4% BSA in PBS for 10 minutes at room temperature. Cells were washed with 0.1% Tween 20 in PBS (PBS-T) and blocked with 10% heat-inactivated goat serum in PBS (blocking buffer) for 1 h at room temperature. Finally, cells were stained with primary Abs (2 µg mL -1 of MR5D3, or anti-EEA1 or anti-LAMP1 diluted in blocking buffer) by incubation for 1 h at room temperature. Subsequently, cells were washed three times with 1 mL PBS-T and incubated with Alexa fluor ® 647 conjugated secondary antibody (1:1000 diluted in blocking buffer, goat anti-Rat for CD206, goat anti-Rabbit for EEA1 or LAMP1) for an additional 60 min at room temperature. Cells were washed as described above and nuclei were stained with 1 µg mL -1 HOECHST solution in PBS for 15 min at room temperature.
In both sets of experiments, after the last cycle of washing, coverslips were mounted with DAKO fluorescent mounting medium and viewed by confocal microscopy using a Zeiss LSM 700 microscope (Carl Zeiss, Heidelberg, Germany) and ZEN2.3 (blue edition) software for images elaboration. Unstained control CD206 + -CHO cells were analyzed to assess cells autofluorescence. Anti-Rat or Anti-Rabbit secondary antibodies concentrations were optimized using a fluorescence microscope to avoid unspecific staining.
A 63x oil objective was used for acquiring all images. Section images in the z-dimension were collected. The quantitative colocalization analysis was performed by using ZEN 2010 confocal microscopy software (Carl Zeiss). Analysis was carried out on the best optical section in the zstack and in sections above and below this plane. Singles cells in 4 different fields were compared for each analysis (12 cells analyzed per sample). The experiments were performed in duplicate.

Resonance (SPR) analysis
Binding experiments were performed by Surface Plasmon Resonance (SPR) on a BIAcore 3000 (Biacore Life Science). Approximately 1300-2000 response units (RU) of soluble CD206 (sCD206) was immobilised on a CM-5 sensor chip surface by amine coupling, to achieve a Rmax of approximately 100 RU during kinetic binding experiments (2).
Where "Rmax" is the maximum analyte binding capacity of the surface, expressed in RU, "analyte MW" is the analyte molecular weight (molecular weight of polymer sugar repeating units), "ligand MW" is the molecular weight of the ligand (soluble CD206, sCD206) and "Rligand" is the signal measured for the amount of immobilised protein in RU.   animals underwent heart perfusion with 60 mL PBS. Heart perfusion was performed to wash out the blood from the liver to eliminate TR-gelatin and glycopolymers still in circulation.

Quantification and imaging of Glycopolymers and TR-gelatin in liver
One liver lobe was accurately weighted, cut in small pieces with the help of scissors and pulverized with a mortar and pestle in liquid nitrogen. The homogenate was resuspended in  100 µL of PBS, or S187 or G187 solution in PBS at a polymer concentration of 400 µM were administered i.p. to mice. After 4 h, 100 µL of a 1.0 mg mL -1 TR-gelatin solution in PBS (treatment) were administered i.p., and liver tissue was collected after further 2 h. b) Due to high fluorescence readingsin the liver several known receptors bind to galactose-containing ligands [16][17][18] -samples were diluted 5:1 vol:vol. The value shown in the table is obtained by multiplying the resulting average fluorescence reading by five: (FI/mg of tissue)*5. While variation of fluorescence may not always follow a perfectly linear trend, this value still provides an indication of higher uptake of galactose-containing polymer G187 when compared to its galactose 3-O-sulfated analogue S187. Figure S15. Representative two-dimensional confocal laser scanning images of liver sections of mice treated with S187, G187 (both 100 µL of 400 µM solutions, polymer concentration, in PBS), or PBS solutions (i.p. administration, 4 h), followed by i.p. injection of 100 µL of Texas Red-tagged gelatin solution in PBS (56 mg kg -1 ). Samples were imaged with Hoechst staining for nuclei (cyan), immunostaining for (A) CD206 or (B) CD31 receptor (red), TR-gelatin (magenta), and S187 or G187 glycopolymers (green) in mouse livers acquired with a 63x immersion oil objective and a magnification of 0.5x. Mice were treated with PBS only (PBS), TR-gelatin only (TR-Gel) or S187 or G187 followed by TR-gelatin (S187 +TR-Gel, or G187 + TR-Gel) as described above. Scale Bar: 20 µm. Figure S16. Representative two-dimensional confocal laser scanning images of liver sections of mice treated with S187, G187 (both 100 µL of 400 µM solutions, polymer concentration, in PBS), or PBS solutions (i.p. administration, 4 h), followed by i.p. injection of 100 µL of TRgelatin solution in PBS (56 mg kg -1 ). Samples were imaged with Hoechst staining for nuclei (cyan), immunostaining for CD31 receptor (red), TR-gelatin (magenta), and S187 or G187 glycopolymers (green) in mouse livers acquired with a 63x immersion oil objective and a magnification of 2x. Mice were treated with PBS only (PBS), TR-gelatin only (TR-Gel), or S187 or G187 followed by TR-gelatin (S187 +TR-Gel, or G187 + TR-Gel) as described above. Scale Bar: 5 µm. Liver sections stained for CD206 are shown in Figure 7.  Figure S17. Representative two-dimensional confocal laser scanning and bright field images of liver sections of mice treated with S187 or G187 (both 100 µL of 400 µM solutions, polymer concentration, in PBS) or PBS solutions (i.p. administration, 4 hours), followed by i.p. injection of 100 µL of TR-gelatin solution in PBS (56 mg kg -1 ). Samples were imaged with Hoechst staining for nuclei (cyan), immunostaining for CD206 receptor (red), TR-gelatin (magenta), Bright Field (BF, gray), and S187 glycopolymers (green) in mouse livers acquired with a 63x immersion oil objective and a magnification of 2x. Mice were treated with PBS only (PBS) or S187 or G187 followed by TR-gelatin (S187 +TR-Gel) as described above. Scale Bar: 5 µm.

Mouse plasma collection and analysis post-treatment with Texas Red-tagged gelatin and glycopolymers
Blood samples (0.7-1 mL) were divided into 2 aliquots, diluted with 1 mL of PBS each and centrifuged at 1,100 rpm for 10 min at 4°C. Supernatants containing plasma fraction were collected and analyzed by spectrofluorometer to assess gelatin (Texas Red) and glycopolymer (Oregon Green) content, recording the emission spectra from 610 to 700 nm (λex = 596 nm) for TR-gelatin, and from 515 to 700 nm (λex = 495 nm) for Oregon Green-tagged glycopolymers G187 and S187. ). (a) Comparison of TR-gelatin plasma levels in mice pre-treated with PBS or S187; (b) comparison of TR-gelatin plasma levels for mice pre-treated with PBS or G187; (c) comparison of glycopolymer-Oregon Green levels for mice pre-treated with PBS or S187; (d) comparison of glycopolymer-Oregon Green levels for mice pre-treated with PBS or G187. For c and d, samples were diluted 1:2 with PBS before analysis as glycopolymer samples were out of range for Oregon Green detection. Data are reported as percentage mean ± s.d. of the fluorescence intensity (the higher value of each group of experiments was set as 100%), N=2 independent biological samples with two technical replicates.

Statistical analysis
Statistical analysis was performed using Prism software 7.04 (GraphPad). Normality was assessed by Shapiro-Wilk normality test. Statistical significance for two group comparison was calculated by two-tailed Student's t-test while for comparison with more than two groups ordinary one-way ANOVA was used. Data are presented as mean values and s.d. Two groups were considered to be significantly different if P<0.05. For the in vivo study, GPower 3.1 was used to perform calculations on sample size. The minimal significance (α) and statistical power (1 − β) were set at 0.05 and 0.80 respectively. Calculations were performed for two groups.